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PubMed1979

Studies on the competition of polyene antibiotics for sterols.

The fractional change in the corrected fluorescence of pimaricin or filipin in the presence of a limiting amount of sterol and a competing polyene antibiotic has been used to estimate the relative affinity of amphotericin B, nystatin, filipin, and pimaricin for stigmasterol and for cholesterol. The relative affinities for cholesterol were filipin greater than amphotericin B greater than pimaricin greater than nystatin, while the relative affinities for stigmasterol were filipin greater than pimaricin greater than amphotericin B greater than nystatin. The data indicate that pimaricin and filipin can both interact simultaneously with about 30% of the cholesterol or stigmasterol. However, the stoichiometry of filipin and pimaricin alone for cholesterol and for stigmasterol in dilute aqueous solutions is 1 : 1. In the experiments which indicated both pimaricin and filipin interact with the same sterol molecule changes in corrected fluorescence and the absorbance spectra were monitored; and these criteria indicated that both pimaricin and filipin had interacted with the sterols. Light-scattering measurements indicate large aggregates were not formed. Although the data shows in dilute aqueous solutions the stoichiometry of filipin and/or pimaricin for sterols is 1 : 1, in more complex solutions, other combinations or interactions are indicated especially for pimaricin.

Amphotericin B↗
PubMed1983

Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol.

We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin-binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze-fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested higher cholesterol in the inner segment than in the outer segment plasma membrane may help direct newly inserted photopigment molecules to the outer segment.

Animals↗
PubMed1986

Plasmalemmal vesicles and the effects of sterol-binding agents in rabbit aortic endothelium.

The rims of vesicle openings in capillary endothelium are reported to be highly sensitive to perturbation by the sterol probe filipin when this agent is administered by perfusion at 50 microM concentration in aldehyde fixative for 10 min. If, as supposed, this specific rim response reflects functionally significant aspects of membrane organization at the vesicle-plasmalemma boundary, then it would be expected to be a reproducible and universal feature of endothelial vesicles in general. In the present study, we have investigated in detail the response to filipin of rabbit and rat aortic endothelial plasmalemma. First, the effect of standard filipin treatment (150 microM, 3-20 h) was examined by freeze-fracture. The extensive generalized response observed, taken together with the effects of filipin seen by fluorescence microscopy of stripped aortic endothelial sheets, and the marked corrugation of the plasmalemma observed by freeze-fracture after tomatin treatment, indicate that substantial amounts of cholesterol are present in the membrane. Exposure of rabbit aortic endothelium to 50 microM filipin for 10, 20, 40 and 60 min enabled the progressive appearance of filipin deformations in the plasmalemma to be traced. Even at the very earliest stages of the response, there was no preferential association of filipin deformations with endothelial vesicle rims. Similar experiments in the rat gave the same result. Specific sensitivity of endothelial vesicle rims would, therefore, seem to be either non-universal in occurrence or non-reproducible, and so is unlikely to reflect any membrane property of fundamental significance to endothelial vesicle function.

Animals↗
PubMed1988

Regional membrane heterogeneity in premyelinated CNS axons: factors influencing the binding of sterol-specific probes.

Binding of the sterol-specific probe filipin to developing optic nerve axonal membrane is spatially heterogeneous prior to association of glial cells with the axons. Experiments were performed using different sterol binding probes (filipin, tomatin, and saponin), at different temperatures (4 degrees C, 23 degrees C, and 37 degrees C), after incubation in different ionic conditions (10 mM Ca2+, 10 mM EGTA, and 20 mM Mg2+), to examine factors that may be responsible for this membrane heterogeneity in rat optic nerve. The patchy pattern of filipin binding is apparent with each sterol-specific probe, even prior to glial ensheathment, and is retained when membrane fluidity is increased at higher temperatures. Increased Ca2+ concentration increased membrane stability, and increased Mg2+ reduced the patchiness of filipin binding. After tannic acid staining, regions of the cytoskeleton are seen associated with the membrane via filaments extending from microtubules to the membrane, preferentially in regions where filipin interaction with the membrane is inhibited. The non-uniform interaction of filipin with the axolemma suggests an underlying heterogeneity in the sterol composition and stability of the membrane. Heterogeneity of premyelinated axonal membrane may provide an important formative influence in the differentiation of axons to their mature morphology and function.

Animals↗
PubMed1985

The tight junction as a barrier to cholesterol in canine epithelial cells.

Filipin has been used to test several models of continuity or flow of lipid components through the tight junction. Cultured canine kidney cells (MDCK) were fixed and incubated in the presence of filipin. Freeze-fracture replicas were analyzed and densities of filipin-cholesterol complexes measured. Fractures of membranes linked with tight junctions were compared statistically to determine whether filipin-cholesterol complexes (protrusions and pits, independently) were randomly distributed between the two membranes of cells separated by the tight junction. The results indicate that filipin-cholesterol complexes are not randomly distributed across the tight junction. If the density of filipin-cholesterol complexes is an accurate indication of membrane cholesterol concentration, then there is a difference in the cholesterol concentration between leaflets of membranes joined by tight junctions and models of the tight junction which suggest leaflet continuity across the junction are in error.

Animals↗
PubMed2003

Accumulation of cholesterol in the lesions of focal segmental glomerulosclerosis.

Intraglomerular deposition of low-density lipoprotein (LDL) and oxidized LDL has been described in various human glomerular diseases. Yet it is not clear whether esterified cholesterol (EC) and unesterified cholesterol (UC) carried in LDL are mobilized from deposited LDL particles or accumulate in the diseased human glomeruli, particularly in the segmentally sclerotic lesions. To address this issue, frozen sections of renal biopsies were first immunostained to localize apolipoprotein B (apo B) and then oil red O (ORO) stained to colocalize neutral lipids. By using 124 ORO-positive biopsies and nine ORO-negative ones, UC was visualized directly with filipin staining, and EC was visualized after its enzymatic hydrolysis and staining with filipin. Seventy-seven biopsies (58%) showed filipin staining of accumulated EC and/or UC in the glomeruli. Of these, 11 showed heavy filipin staining for both EC and UC in the segmentally sclerotic lesions. In a group with UC deposits in the sclerotic segments, the percentage of the glomeruli affected by sclerosis and the intensity of filipin fluorescence for UC were significantly higher than biopsies with only mesangial UC deposits. Most filipin-positive biopsies showed apo B staining mainly in the mesangium. Yet in the sclerotic segments, apo B staining was rarely noted. Accumulated apo B-stained lipoprotein was not coincident with ORO-stained lipid in the diseased glomeruli. These results suggest that both EC and UC accumulate in the sclerotic glomerular segments as the glomerular lesions are advanced, and that these EC and UC appear to be derived from altered LDL with progressive loss of apo B.

Adult↗
PubMed1982

Distribution of cytochemically detectable cholesterol in the electric organ of Torpedo marmorata.

A cytochemical probe for cholesterol, the polyene antibiotic filipin, was applied to aldehyde-fixed samples of the electric organ of Torpedo marmorata to identify filipin-binding sites in the various membrane components of the organ and, hence, the probable cholesterol content at these levels. In both thin-sectioned and freeze-fractured samples, filipin-cholesterol complexes appeared numerous and homogeneously distributed on the Schwann cell plasma membrane. On the presynaptic membrane, filipin-cholesterol complexes occurred in patches alternating with unlabeled membrane segments. The postsynaptic, acetylcholine receptor-rich plasma membrane of the electroplax showed no or few filipin-cholesterol complexes in the flat region and upper part of the invaginations (both areas characterized by a lattice of small intramembrane particles); however, the membrane of the bottom part of the postsynaptic invaginations contained several complexes. The ventral, noninnervated plasma membrane of the electroplax showed a moderate, homogeneous filipin labeling. These data suggest that the distribution of cholesterol among membranes of the electroplax is not homogeneous and that the acetylcholine receptor-rich region of the postsynaptic membrane (as characterized by the lattice of small intramembrane particles) may contain little cholesterol.

Animals↗
PubMed1983

Distribution of sterol-specific complexes in a continually shearing region of a plasma membrane and at procaryotic-eucaryotic cell junctions.

A narrow zone of plasma membrane between the head and body of a protozoan from termites undergoes continual in-plane shear because the head rotates continuously in the same direction relative to the cell body (Tamm, S.L., and S. Tamm, 1974, Proc. Natl. Acad. Sci. USA 71:4589-4593). Using filipin and digitonin as cytochemical probes for cholesterol and related 3-beta-hydroxysterols, we found a high level of sterol-specific complexes, visible as membrane lesions in thin sections, in both shearing and nonshearing regions of the membrane, indicating no difference in sterol content. This confirmed previous observations that any region of the fluid membrane can undergo shear, but that this occurs only at certain locations due to cell geometry and proximity to rotating cytoskeletal structures. Filipin and digitonin did not disrupt the plasma membrane at the junctions with ectosymbiotic rod and fusiform bacteria (i.e., membrane pockets and ridges). However, pepsin degradation of dense material coating the junctional membranes resulted in a positive response of these regions to filipin. Fluorescence microscopy revealed a bright halo around each rod bacterium, due to filipin-sterol binding in the sides of the membrane pockets, but no fluorescence at the bottom of the pockets; the same fluorescence pattern was found in pepsin-treated cells despite the presence of sterols throughout the pocket membrane, as shown by electron microscopy. These findings indicate that (a) regional constraints may restrict the ability of filipin to interact with sterols or form visible membrane lesions, and (b) a negative response to filipin, assayed by either electron or fluorescence microscopy, is not sufficient to demonstrate low membrane sterol concentration, particularly in membrane domains characterized by closely associated proteins.

Animals↗
PubMed1983

Rings of membrane sterols surround the openings of vesicles and fenestrae, in capillary endothelium.

We investigated the distribution of sterols in the cell membrane of microvascular endothelium (mouse pancreas, diaphragm, brain, heart, lung, kidney, thyroid, adrenal, and liver) with the polyene antibiotic filipin, which reportedly has binding specificity for free 3-beta-hydroxysterols. In some experiments, concomitantly, cell-surface anionic sites were detected with cationized ferritin. Vessels were perfused in situ with PBS, followed by light fixation and filipin administration for 10 to 60 min. Tissues were further processed for thin-section and freeze-fracture electron microscopy. Short exposure (10 min) to filipin-glutaraldehyde solution resulted in the initial appearance, on many areas, of rings of characteristic filipin-sterol complexes within the rim surrounding stomata of most plasmalemmal vesicles, transendothelial channels, and fenestrae. Such rings were absent from the rims of the large openings of the sinusoid endothelium (liver, adrenal), coated pits and phagocytic vacuoles. After longer exposure (30-60 min), filipin-sterol complexes labeled randomly the rest of plasma membrane (except for coated pits, and partially the interstrand areas of junctions), and also marked most plasmalemmal vesicles. These peristomal rings of sterols were displayed mostly on the P face, and, at their full development, consisted of 6-8 units around a vesicle stoma, and 10-12 units around a fenestra. At their level, the intramembranous particles and the cell surface anionic sites were virtually excluded. Peristomal rings of sterols were also detected on the plasma membrane of pericytes and smooth muscle cells of the microvascular wall, which otherwise were poorly labeled with filipin-sterol complexes as compared to endothelial plasmalemma. It is presumed that the peristomal rings of cholesterol may represent important contributors to the local transient stabilization of plasma membrane and to the phase separation between cell membrane and vesicle membrane at a certain stage of their fusion/fission process.

Animals↗
PubMed1982

A correlative study on the topographical distribution of the receptors for low density lipoprotein (LDL) conjugated to colloid gold in cultured human skin fibroblasts employing thin section, freeze-fracture, deep-etching, and surface replication techniques.

The topographical distribution of the receptors for low-density lipoprotein (LDL) conjugated to colloidal gold on cultured human skin fibroblasts was studied using a surface replication technique following critical point drying. In this correlative study, also employing thin sectioning, freeze-fracture and deep-etching techniques in conjunction with the polyene antibiotic filipin, it could be shown that LDL preferentially binds to certain microdomains of the plasma membrane, the so-called coated pits. The coated pits can be recognized after filipin treatment both on the P face and E face of the membrane, due to the fact that they are free of filipin-sterol complexes. Filipin was also of value in clearly delimiting the structure of nascent coated pits from their surroundings in surface replicas. Using filipin has the effect of increasing the contrast between the smooth surface of a coated pit and the rough surface of the remaining membrane caused by formation of filipin-sterol complexes. This study has shown that there are differences in the topographical distribution and number of gold particles from fibroblast to fibroblast and even between different areas of the same cell. The surface replication technique using goldlabelled LDL provides s suitable method for improving the interpretation of the spatial arrangement of the LDL receptors. The advantages of this technique are discussed in comparison with the methods previously employed to visualize LDL receptors.

Cell Membrane↗
PubMed1979

Control of permeation of bleomycin A2 by polyene antibiotics in cultured Chinese hamster cells.

Control of permeation of bleomycin A2, a well-known antitumor antibiotic, in combination with various polyene macrolide antibiotics was analyzed in cultured Chinese hamster cells in vitro. Three polyene antibiotics, filipin, pentamycin, and pimaricin, were found to enhance the action of bleomycin A2 remarkably, while amphotericin B or nystatin could not. Although DNA synthesis and colony-forming activity of polyene-sensitive Chinese hamster V79 cells were synergistically inhibited by the combination of filipin and bleomycin A2, in a polyene-resistant subline (AMBR-1) derived from V79, they were only slightly affected in the presence of both drugs. The cellular uptake of [14C]bleomycin A2 by V79 was enhanced 2- to 4-fold in the presence of increasing doses of filipin or pentamycin, but not in the presence of amphotericin B. The treatment of V79 cells with filipin for 20 to 30 min was enought to block DNA synthesis almost completely when combined with 20 microgram belomycin A2 per ml. The pretreatment of the hamster cells with 6 microgram filipin per ml for 60 min continued to enhance the inhibitory action by bleomycin A2 of DNA synthesis up to 5 hr after the removal of filipin from the cultured medium.

Amphotericin B↗
PubMed1982

Cholesterol distribution and structural differentiation in the sarcoplasmic reticulum of rat cardiac muscle cells. A freeze-fracture cytochemical investigation.

The polyene compound, filipin, was used as a probe to localize cholesterol in the membranes of the rat cardiac muscle cell, with particular reference to the sarcoplasmic reticulum (SR). Filipin binds specifically to cholesterol (and related 3-beta-hydroxysterols) in membranes, producing distinct deformations which can be viewed by freeze-fracture and used as markers for the presence of cholesterol-rich regions in the membrane plane. In freeze-fracture replicas of filipin-treated rat myocardium, the muscle cells revealed abundant deformations in their plasma membranes, no deformations in mitochondrial membranes, and an intermediate response in the SR. These results are in agreement with the levels of cholesterol reported in isolated fractions of the different membrane types, and confirm the specificity of filipin action. Within the SR, the filipin-induced deformations were not randomly distributed but occurred more commonly in free SR at or near the Z-region of the sarcomere than in other parts of the free SR or the junctional SR. This finding is interpreted as evidence for a non-homogeneous distribution of cholesterol in cardiac muscle cell SR. The possible significance of cholesterol in relation to structural differentiation and function of the SR is discussed.

Animals↗
PubMed1984

Cytochemical characteristics of astrocytic plasma membranes specialized with numerous orthogonal arrays.

Astrocytic membranes contacting the basal lamina are found to be less affected by filipin than subjacent lateral membranes. An abrupt change in density of lesions induced by filipin creates a border between subpial and lateral membranes at the glia limitans. This means that orthogonal array-crowded membranes may contain relatively less cholesterol than other astrocytic membrane domains. Another possible explanation for filipin resistance is also considered in relation to aggregated intramembrane particles of orthogonal arrays and/or membrane-associated filamentous elements including the basal lamina. The polygonal particle junction between astrocytic processes located just below the subpial membrane is strongly resistant to the action of filipin. Both membrane-associated enzymes, i.e. alkaline phosphatase (AlkPase) and Na+,K+-ATPase are commonly detected only in perivascular astrocytic membranes, and not in subpial membranes, suggesting a regional differentiation in function of astrocytic membranes. There are variations in the reactive deposits particularly of those for Na+,K+-ATPase. It is apparent that the distribution polarity of orthogonal arrays is not connected with that of either AlkPase or Na+,K+-ATPase. Judging from the relative resistance to filipin, however, astrocytes throughout the C.N.S., having domains specialized with orthogonal arrays, may possess a unique stabilizing mechanism for their own membranes contacting the basal lamina.

Alkaline Phosphatase↗